The goal of this project is to provide a comprehensive view of the genetic organization and metabolic capabilities of Treponema pallidum and to correlate this information with the unique physiology of this bacterium and its pathogenesis in syphilis and other treponematoses. Syphilis continues to be a major health problem in the United States, as indicated by a 53% increase in the reported incidence of primary and secondary syphilis over the past three years. Study of T. pallidum subsp. pallidum (the causative agent of syphilis) and other subspecies causing yaws and endemic syphilis has been hampered by the inability to culture these spirochetes continuously in vitro. Because clonal populations can not be selected and propagated, it has not been possible to examine the genetics of T. pallidum by standard methods such as mutation, transformation, conjugation, and transduction. As a result, less is known about the genetic organization of T pallidum than that of any other human pathogen. We have already determined that T. pallidum has an extremely small genome (about 900 kilobase pairs). In this project, a physical map of the T pallidum genome will be constructed using pulsed field gel electrophoresis and rare-cutting restriction enzymes, and an ordered cosmid library will be established to facilitate further genetic analysis. Cosmid libraries will be used in combination with an extensive collection of E. coli auxotrophs to survey the presence of important metabolic genes in T pallidum by gene complementation, thereby refining our knowledge of the physiologic capabilities of the organism. The T pallidum genes identified in this project or previously characterized by other investigators will be localized within the physical map by procedures using RecA-directed site-specific cleavage or transposon mutagenesis of complementing clones. Finally, the genetic content of different T. pallidum strains, subspecies, and other treponemal species will be compared in an effort to understand the underlying differences in physiology and pathogenesis. Development of a physical and genetic map of T. pallidum and the other studies described here will provide a basic framework for future genetic studies and will shed light on the unusual pattern of pathogenesis and physiologic properties of this group of organisms.